Ribosome-mediated synthesis of proteins begins with a methionine residue. In prokaryotes and eukaryotic organelles (mitochondria and chloroplasts), the methionyl moiety carried by the initiator tRNA is N-formylated prior to its incorporation into a polypeptide (Meinnel and Blanquet, J. Bacteriol. 175:7737-7740 (1993)). N-formylmethionine is therefore always incorporated at the N-terminus of a nascent polypeptide in prokaryotes (Adams, J. M. and Capecchi, M., Proc. Natl. Aca. Sci. U.S.A. 55:147-155 (1966); Webster et al., Proc. Natl. Acad. Sci. U.S.A. 55:155-161 (1966)). However, most mature proteins do not retain the N-formyl group (Marcker, K. and Sanger, F. J. Mol. Biol. 8:835-840 (1964)). Instead, the N-formylmethionine group is removed post- or co-translationally in a process known as deformylation, which is catalyzed by peptide deformylase.
Deformylation is catalyzed by peptide deformylase which cleaves the formyl group from the nascent polypeptide chain (Adams, J., J. Mol. Biol. 33:571-589 (1968); Livingston, D. M. and Leder, P., Biochemistry 8:435-443 (1969); Takeda, M. and Webster, R. E., Proc. Natl. Acad. Sci. U.S.A. 60:1487-1494 (1968)).
Prior to the present invention, research in the area of peptide deformylase activity and enzymology focused on the bacterial enzyme. For example, the structure of the core domain of E. coli peptide deformylase was solved by NMR (Meinnel, T. et al., J. Mol. Biol. 262:375-386 (1996)) and the structure of the full-length protein by X-ray crystallography (Chan et al., Biochemistry 36:13904-13909 (1997)). Becker et al., J. Biol. Chemistry 273(19): 11413-11416 (1998) solved the structure of the catalytically active E. coli enzyme in the nickel-bound form (PDF-Ni) and in inhibitor-complexed form.
More recently, Ragusa et al., J. Mol. Biol. 289: 1445-1457 (1999), investigated the substrate specificity of Escherichia coli peptide deformylase by measuring the efficiency of the enzyme to cleave formylpeptides. Durand et al., Archives Biochemistry and Biophysics, 367(2):297-302 (1999), tested a variety of peptide aldehydes and identified calpeptin as a potent inhibitor of E. coli and B. subtilis peptide deformylase.
The overall focus in the field has been engineering site-specific inhibitors of peptide deformylase with a goal of developing broad-spectrum antibiotics with low to no mammalian toxicity. Rajagopalan et al., Biochemistry 36:13910-13918 (1997), identified specific and potent inhibitors of the bacterial enzyme which are potentially novel and new antibiotic agents. Meinnel et al., Biochemistry 38:4287-4295 (1999) designed and synthesized substrate analogue inhibitors of bacterial peptide deformylase and studied their capacity to undergo hydrolysis. To aid in the design of both the cobalt and zinc containing E. coli peptide deformylase, Hao et al., Biochemistry 38:4712-4719 (1999), studied the structure of the protein-inhibitor complexes of the cobalt and zinc containing E. coli enzyme.
Prior to the present invention, it was generally accepted that peptide deformylase genes and proteins were absent from eukaryotic cells. The present invention unexpectedly establishes, for the first time, the existence of a peptide deformylase gene and protein in eukaryotic cells, particularly in plant cells. The inventors have found that the deformylase is a novel and suitable target for identifying new broad spectrum herbicides.